Design and Synthesis of Some New 1,2,4-Triazolo[4,3-a]Quinoxaline Derivatives as Potential Antimicrobialagents

As a part of an ongoing research program to achieve new chemical entities suitable for development as new class of antimicrobial agents, the present work describes the design and synthesis of a new series of substituted-1-methyl-1,2,4-triazolo[4,3-a]quinoxalines, The newly synthesized compounds were screened for their in vitro antimicrobial activity. The results revealed that the compounds demonstrated significant activity against Gram negative bacteria. Compounds 3 and 11b exhibited twice the activity of ampicillin against Pseudomonas aeruginosa, while compounds 4, 5b, 7, 9a, 10d, 11a, 11c and 12 were equipotent to ampicillin. On the other hand, the tested compounds demonstrated mild antifungal activity. Compound 11d exhibited nearly one-half the activity of clotrimazole against Candida albicans.


Introduction
Resistance of pathogenic bacteria towards the clinically used antibiotics makes it harder to eliminate infections from the body as existing drugs become less effective creating a challenging problem worldwide. As a result, discovery and development of new class of antimicrobial drugs are urgently needed to combat the growing threat of drug-resistant microbes [1].
Literature survey revealed that quinoxaline and fused quinoxaline ring systems are attractive candidates in medicinal chemistry as they constitute the building blocks of wide range of many pharmacologically active compounds having anticancer [2], antimicrobial [3,4], antiinflammatory [5], antidepressant [6], antiviral [7], antidiabetic [8], antihypertensive [9], antihistaminic [10] and antiglaucoma activities [11]. In addition, it has been reported that quinoxaline moiety constitutes the basic skeleton for many natural and synthetic pharmacologically active compounds [12]. For example, quinoxaline ring is a part of the naturally occurring antibiotics, triostin A and echinomycin that are known to inhibit the growth of Gram positive bacteria and they are active against various transplantable tumors [13][14][15] Moreover, in a previous publication, the synthesis and antimicrobial evaluation of a series of substituted 1,2,4-triazolo [4,3-a]quinoxalines have been reported [16,17]. The screening results revealed that compounds A and B ( Figure 1) exhibited significant activity against Staphylococcus aureus and Candida albicans respectively.
In view of the above mentioned results and as a continuation of our research on quinoxaline derivatives in an attempt to identify new lead compounds that might be of value for future development as new class of antimicrobial agents, we report herein the synthesis and antimicrobial evaluation of a new series of 5-substituted 1,2,4triazolo [4,3-a]quinoxaline derivatives (formula A, Figure 2) in order to achieve further knowledge of the structure-activity relationship. the microanalyses were within ± 0.4 % of the calculated values. Followup of the reactions and checking the homogeneity of the compounds were made by ascending TLC run on silica gel G (Merck 60) coated glass plates. The spots were visualized by exposure to iodine vapor or UV lamp at k 254 nm for few seconds. Compound 1 was prepared according to the reported method [19] by refluxing 1-methyl-1,2,4triazolo [4,3-a]quinoxalin-4(5H)-one with acetic anhydride . Heating 1 with phosphorous oxychloride afforded compound 2 according to a previously reported reaction conditions [20][21][22].

Experimental Chemistry
All reagents and solvents were purchased from commercial suppliers and were dried and purified when necessary by standard techniques. Melting points were determined in open glass capillaries using Stuart capillary melting point apparatus (Stuart Scientific Stone, Staffordshire, UK) and are uncorrected. IR spectra were recorded, for potassium bromide discs, ύ (cm -1 ), on Perkin Elmer 1430 spectrophotometer. 1 H-NMR spectra were determined either on a Bruker Avance spectrometer (300 MHz) at the microanalytical unit, Faculty of Science, Cairo University, or on Jeol (500 MHz) at the microanalytical unit, Faculty of Science, Alexandria University, using DMSO-d6 as a solvent and TMS as internal standard. The chemical shifts are given in δ ppm values (s, singlet; d, doublet; t, triplet and m, multiplet). 13

6-Methyltetrazolo[1,5-a]-1,2,4-triazolo[3,4-c]quinoxaline (12):
An ice-cold solution of sodium nitrite (0.07 gm, 1 mmol) in water (2 ml) was added dropwise to a stirred solution of 3 (0.21 gm, 1 mmol) in hydrochloric acid (1-2 ml). The reaction mixture was stirred at room temperature for 3 h during which precipitation of white product occurred. The obtained product was filtered, washed with water, dried and crystallized from dimethylformamide.   On the other hand, condensation of 3 with the appropriate aromatic aldehyde in boiling ethanol afforded the corresponding hydrazones 10a-d. 1 H-NMR data confirmed the existence of the two geometrical isomers E and Z of compounds 10a-c as it revealed the existence of two upfield singlets assigned to two CH 3 groups of the two isomers and two deshielded D 2 O exchangeable singlets corresponding to the NH groups, in addition to the aromatic signals integrated to the double number of triazoloquinoxaline protons. The 1 H-NMR spectrum of 10c characterized by the existence of two upfield singlets assigned for the protons of the OCH 3 groups. It is worthy to mention that the ratio of the paired signals corresponding to the two geometric isomers is 2:1. On the other hand, the 1 H-NMR spectrum for 10d did not show paired signals for any protons which could be explained by steric hindrance of the benzodioxole moiety that force the molecule to exist in the most stable isomer. Compouds 10a-d underwent oxidative cyclization by bromine in presence of anhydrous sodium carbonate to the corresponding bis-triazoloquinoxalines 11ad. 1 H-NMR spectra for 11a-d revealed the disappearance of the two singlets corresponding to N=CH and NH protons present in their precursors. 1 H-NMR spectra for 11a-c lacked the paired signals for CH 3 and triazoloquinoxaline protons which confirms the disappearance of the E and Z geometrical isomers by cyclization. Moreover, reacting 3 with sodium nitrite solution in hydrochloric acid at 5°C gave the target tetrazolo derivative 12. The structures of the newly synthesized compounds were substantiated by elemental analyses, IR, MS, 1 H-NMR and 13 C-NMR spectral data (experimental section).

Biological evaluation
Antimicrobial screening: All the newly synthesized compounds were preliminary evaluated for their in-vitro antibacterial activity against Staphylococcus aureus and Bacillus subtilis as Gram-positive bacteria, Escherichia coli and Pseudomonas aeruginosa as Gramnegative bacteria. They were also tested for their in-vitro antifungal potential against Candida albicans. Their inhibition zones using the cup-diffusion technique [23] were measured and further evaluation was carried out to determine their minimal inhibitory concentration (MIC) and minimum bacterial concentration (MBC) using the twofold serial dilution method [24]. Ampicillin was used as standard antibacterial while clotrimazole was used as antifungal reference. Dimethylsulfoxide (DMSO) was used as blank and showed no antimicrobial activity.
As revealed from tables 1 and 2, the tested compounds displayed promising inhibitory effects on the growth of the tested organisms. In general, the compounds were highly effective against Gram-negative bacteria than Gram-positive and fungi. Compounds 3 and 11b proved to be two times as active as ampicillin (MIC = 25 µg/mL) against P. aeruginosa. Whereas, compounds 4, 5b, 7, 10d, 11a, 11c and 12 were as active as the reference. While, compounds 3, 4, 5b, 8a, 9a, 10a and 12 (MIC = 25 µg /mL) showed nearly half the activity of ampicillin against E. coli.
On the other hand, the results revealed that the tested compounds displayed notable antifungal activity. Compound 11d exhibited nearly one-half the activity of clotrimazole against C. albicans. (MIC = 12.5 µg/mL).
According to the MIC and MBC limits derived from the latest National Committee on Clinical Laboratory Standards (NCCLS), it can be determined whether the test compound is bactericidal or bacteriostatic to the test organism. Accordingly, and as revealed from table 2, only compounds 10d and 11c were bactericidal against P. aeruginosa while the remaining compounds were bacteriostatic against the test organisms.
Structural-activity correlation of the tested compounds indicated that 5-substituted-1-methyl-1,2,4-triazoloquinoxalines (4 and 5b)  demonstrated promising activity against P. aeruginosa, being as active as ampicillin. Moreover, they displayed notable activity against E. coli. While, the 4-hydrazinyl-1-methyl-1,2,4-triazoloquinoxaline 3 showed enhanced activity towards P. aeruginosa, being two time as active as the reference which might be due to the presence of 4-hydrazino group which increased the possibility of hydrogen bonding. Conversion of 3 into the corresponding Schiff 's bases 10a-c resulted in remarkable decrease in activity against P. aeruginosa being one-half as active as the reference. While, derivative 10d was found to be as active as reference against P. aeruginosa. Such activity might be due to the presence of the 1,3-dioxole moiety.
Furthermore, cyclization of compounds 10a-c into 10-aryl-3methylbis-1,2,4-triazoloquinoxalines 11a-c resulted in an increase of activity towards P. aeruginosa. The presence of Cl atom at position-4 of the phenyl ring in 11b enhanced the activity against P. aeruginosa to be twice the activity of the reference. On the other hand, cyclization of compound 10d into the corresponding bistriazolo derivative 11d led to decrease in antibacterial activity against P. aeruginosa.
Cyclocondensation of 3 into the lipophilic tetracyclic bistriazoloquinoxalines 7 and 8a,b decreased the antibacterial activity towards the Gram negative P. aeruginosa which could be explained by the increase of lipophilicity of the cyclic compounds. While cyclocondensation of 3 into the hydrophilic 10-carboxy bistriazolo analog 9a exhibited activity as the reference against P. aeruginosa. It is worthy to mention that the p-nitrophenyl ring in compound 8b might be the reason for increasing the antibacterial activity towards B. subtilis. As well, the enhanced activity of compound 12 towards B.subtilis and E. coli might be attributed to the tetrazole moiety.
Antimicrobial screening results indicated that the target compounds were highly effective against G-negative bacteria than G-positive bacteria and fungi. Compounds 3 and 11b displayed twice the activity of that of the reference ampicillin. Whereas compounds 4, 5b, 7, 9a, 10d, 11a, c and 12 were as active as the reference against P. aeruginosa. Consequently, such series of compounds could be considered as structural leads that deserve further structural modification and investigation to optimize their antimicrobial efficacy aiming at finding out a new class of antimicrobial agents.